Design and development of neurokinin-1 receptor-binding agent delivery conjugates

ABSTRACT

Neurokinin-1 (NK-1) receptor-binding agent delivery conjugates, compositions comprising NK-1 receptor-binding agent delivery conjugates, and methods for making and administering NK-1 receptor-binding agent delivery conjugates are provided. A conjugate may include an NK-1 receptor-binding moiety, a tinker group containing at least one linker selected from the group of a releasable linker and a spacer linker, and an active agent linked to the linker group. The active agent may be selected from the group of fluorophore-containing compounds, radionuclide-containing compounds, and therapeutic agents for treatment of tumor cells characterized by over-expression of the NK-1 receptor.

BACKGROUND

The mammalian immune system provides mechanisms for the recognition andelimination of tumor cells invading foreign pathogens. While the immunesystem normally provides a strong line of defense, tumor cells orpathogens may nonetheless evade a host immune response and proliferateor persist with concomitant host pathogenicity. Chemotherapeutic agentsand radiation therapies have been developed to eliminate, for example,replicating neoplasms. However, many of the currently availablechemotherapeutic agents and radiation therapy regimens have adverse sideeffects by likewise affecting normal host cells, such as cells of thehematopoietic system. The adverse side effects of these anticancer drugshighlight the need for the development of new therapies selective forpathogenic cell populations and with reduced host toxicity. Moreover,the target selectiveness can be implemented to improve other therapeuticand diagnostic techniques that are tailored to a pathogenic cellpopulation.

Researchers have developed therapeutic protocols for destroyingpathogenic cells by targeting cytotoxic compounds to such cells. Many ofthese protocols utilize toxins or chemotherapeutic agents conjugated toantibodies that bind to antigens that are unique to, or over-expressedby, the pathogenic cells in an attempt to minimize delivery of the toxinto normal cells.

The neurokinin-1 (NK-1) receptor (also known as the tachykinin receptor1 or SP receptor) is a G protein-coupled receptor that is the product ofthe TACR1 gene. The NK-1 receptor is 407 amino acid residues, and, alongwith other tachykinin receptors, is made of seven hydrophobictransmembrane domains with three extracellular and three intracellularloops, an amino-terminus, and a cytoplasmic carboxy-terminus. The loopshave functional sites, including two cysteines amino acids for adisulfide bridge, Asp-Arg-Tyr that is responsible for association witharrestin, and Lys/Arg-Lys/Arg-X-X-Lys/Arg, which interacts withG-proteins. While the NK-1 receptor has some affinity for alltachykinins, its endogenous ligand is the peptide Substance P (SP).

Upon binding to the NK-1 receptor, SP has been shown to induce changessuch as tumor cell proliferation, angiogenesis, and migration of thetumor cells for metastasis. In contrast, NK-1 receptor antagonists exertantiproliferative effects on NK-1-receptor expressing cells by inducingapoptosis. Moreover, it is known that NK-1 receptors are overexpressedin some tumors, and that tumor cells express several isoforms of theNK-1 receptor. For example, NK-1 receptor over-expression has beenreported in certain cancers of the larynx, stomach, colon, pancreas, andbreast as well as glioblastomas, gliomas, astrocytomas, melanomas,retinoblastomas, and neuroblastomas.

All of these data suggest that NK-1 receptor expression may play animportant role in the development of cancer, that SP may be a universalmitogen in NK-1 receptor-expressing tumor cells, and/or that expressionof the NK-1 receptor may be utilized to diagnose or identify specifictumors. Further, the data suggest that NK-1 receptor antagonists couldoffer a promising therapeutic strategy for the treatment of humancancer, since they act as broad-spectrum antitumor agents. In sum, theNK-1 receptor may be a new and promising target in the diagnosis and/ortreatment of human cancer.

SUMMARY

The various embodiments provide compositions and methods for making andadministering a neurokinin-1 (NK-1) receptor-binding agent deliveryconjugate that includes an NK-1 receptor-binding moiety, a linker groupthat includes one or more linker selected form the group of a releasablelinker and a spacer linker, and an active agent linked to the linkergroup.

In some embodiments, the linker group includes at least one spacerlinker, and the active agent is selected from the group consisting of afluorophore-containing compound and a radionuclide-containing compound.In some embodiments, the linker group includes at least one releasablelinker, and the active agent is a therapeutic agent. In someembodiments, the active agent is a radionuclide-containing compoundincluding one of technetium-99m and copper-64, and a chelating portionthat forms a complex with the radionuclide.

In some embodiments, the active agent is a therapeutic agent fortreatment of tumor cells characterized by over-expression of the NK-1receptor, in which the therapeutic agent is selected from the group oftubulysin B hydrazide (TubH) and disulfide-activated desacetylvinblastine hydrazide (DAVBH).

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated herein and constitutepart of this specification, illustrate example aspects of the invention,and together with the general description given above and the detaileddescription given below, serve to explain the features of the invention.

FIG. 1A is a schematic reaction equation illustrating the formation ofan NK-1 receptor-binding moiety.

FIG. 1B provides in vivo mice therapeutic data for HEK 293-NK1R tumorxenograft models showing behavior of tumor volumes from the NK1R-EC20peptide linker-TubH conjugate.

FIG. 1C provides in vivo mice therapeutic data on HEK 293-NK1R tumorxenograft models showing behavior body weights during the therapy shownin FIG. 1B.

FIG. 2 is a schematic reaction equation illustrating the synthesis of anNKIRL-Lys peptide-rhodamine conjugate.

FIG. 3 is a schematic reaction equation illustrating the synthesis of anNK1RL-EC20 peptide-LS288-maleimide conjugate.

FIG. 4 is a set of confocal microscopy images showing in vitro bindingof NKIRL-Lys peptide linker-rhodamine to HEK 293-NK1R cells.

FIG. 5 is a graph showing the binding of NKIRL-Lys peptide rhodamine toHEK 293-NK1R cells by flow cytometry.

FIGS. 6A and 6B are graphs showing binding affinity of two fluorescentimaging conjugates in cultured HEK 293-NK1R cells expressing NK-1receptor.

FIG. 6C is a set of images showing (a-b) HEK 293-NK1R tumor xenograftmodel mice treated with conjugates in which LS288 is the active agent,(c-d) blocking images for the HEK 293-NK1R tumor xenograft model micewith the treatment in a-b, and (e-f) NK1R-negative tumor xenograft modelmice treated with conjugates in which LS288 is the active agent.

FIG. 6D is a biodistribution study of the mice imaged in FIG. 6C.

FIG. 7A is a plot showing the radioactivity binding affinity of anNK1RL-EC20 peptide linker-^(99m)Tc conjugate versus concentration of theconjugate in cells.

FIG. 7B is a set of whole body mice images showing HEK 293-NK1R tumorxenograft model treated with the NK1RL-EC20 peptide linker-⁹⁹ ^(m) Tcconjugate (left), and showing blocking (right).

FIG. 7C is a biodistribution study of the imaged mice from FIG. 7B forthe NK1RL-EC20 peptide linker-^(99m)Tc conjugate and a competitive NK-1receptor ligand.

FIG. 7D is a set of whole body mice images on SPECT-CT for NK1RL-EC20peptide linker-⁹⁹ ^(m) Tc conjugate in HEK 293-NK1R tumor xenograftmodel mice.

FIG. 7E is a set of whole body mice images on SPECT-CT for NK1RL-EC20peptide linker-^(99m)Tc conjugate in NK1R-negative tumor xenograft modelmice.

FIG. 8A is a plot showing region of interest (ROI) radioactivity ofNK1RL-PEG2-NOTA-{circumflex over ( )}Cu conjugate in NK1R-transduced andnon-transduced xenografts.

FIG. 8B is a plot showing the NK1RL-PEG2-NOTA-⁶⁴Cu conjugate uptakeratio between the NK1R-transduced and non-transduced xenografts invarious areas at 20 hours post-injection.

FIG. 8C is a set of PET images showing HEK 293-NK1R tumor xenograftmodel mice treated with a conjugate in which the active agent has ⁶⁴Cu.

FIG. 9A is a plot showing the radioactivity binding affinity of anNK1RL-PEG36-based short EC20 linker-”^(m)Tc conjugate versusconcentration of the conjugate in cells in the presence and absence of acompeting ligand.

FIG. 9B is a set of whole body mice images showing HEK 293-NK1R tumorxenograft model treated with the NK1RL-PEG36-based short EC20 linker-⁹⁹^(m) Tc conjugate with shielding (top row), and without shielding(bottom row).

FIG. 9C is a biodistribution study of the imaged mice from FIG. 9B forthe NK1RL-PEG36-based short EC20 linker-^(99m)Tc conjugate and acompetitive NK-1 receptor ligand at 2 hours post-injection.

FIG. 9D is a biodistribution study of the imaged mice from FIG. 9B forthe NK1RL-PEG36-based short EC20 linker-^(99m)Tc conjugate and acompetitive NK-1 receptor ligand at 8 hours post-injection.

FIG. 10A is a schematic reaction equation illustrating the synthesis ofan NK1RL-SF5-Lys peptide linker-rhodamine conjugate.

FIG. 10B is a plot showing the fluorescence binding affinity of anNK1RL-SF5-Lys peptide linker-rhodamine conjugate in cultured HEK293-NK1R cells expressing NK-1 receptor.

FIG. 10C is a set of confocal microscopy images showing in vitro bindingof NK1RL-SF5-Lys peptide linker-rhodamine to HEK 293-NK1R cells.

FIG. 11 is a schematic reaction equation illustrating the synthesis ofan NK1RL-SF5-tyrosine peptide linker-S0456 conjugate.

FIG. 12 is a plot showing the fluorescence binding affinity of anNK1RL-SF5-tyrosine peptide linker-S0456 conjugate versus concentrationof the conjugate in cells in the presence of a competing ligand.

FIG. 13A is a PET image showing a HEK 293-NK1R tumor xenograft modelmouse treated with an NK1RL-PEG2-DOTA-¹ ¹¹ Iη conjugate.

FIG. 13B is a plot showing the NK1RL-PEG2-DOTA-¹ ¹ ¹in conjugate uptakeratio in the HEK 293-NK1R tumor xenograft model in various areas at 4hours post-injection.

FIG. 13C is a whole body mouse image on SPECT-CT for NK1RL-PEG2-DOTA-¹ ¹¹in conjugate in the HEK 293-NK1R tumor xenograft mouse.

DETAILED DESCRIPTION

The present invention may be understood more readily by reference to thefollowing detailed description taken in connection with the accompanyingfigures and examples, which form a part of this disclosure. It is to beunderstood that the various embodiments are not limited to the specificdevices, methods, applications, conditions or parameters describedand/or shown herein, and that the terminology used herein is for thepurpose of describing particular embodiments by way of example only andis not intended to be limiting.

It is to be appreciated that certain features that are, for clarity,described herein in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures that are, for brevity, described in the context of a singleembodiment, may also be provided separately or in any sub-combination.Further, reference to values stated in ranges include each and everyvalue within that range.

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural referents unless the contentclearly dictates otherwise.

The term “alkyl” as used herein refers to a monovalent linear chain ofcarbon atoms that may be optionally branched, such as methyl, ethyl,propyl, 3-methylpentyl, and the like.

The term “cycloalkyl” as used herein refers to a monovalent chain ofcarbon atoms, a portion of which forms a ring, such as cyclopropyl,cyclohexyl, 3-ethylcyclopentyl, and the like.

The term “alkylene” as used herein refers to a bivalent linear chain ofcarbon atoms that may be optionally branched, such as methylene,ethylene, propylene, 3-methylpentylene, and the like.

The term “cycloalkylene” as used herein refers to a bivalent chain ofcarbon atoms, a portion of which forms a ring, such ascycloprop-1,1-diyl, cycloprop-1,2-diyl, cyclohex-1,4-diyl,3-ethylcyclopent-1,2-diyl, 1-methylenecyclohex-4-yl, and the like.

The term “heterocycle” as used herein refers to a monovalent chain ofcarbon and heteroatoms, wherein the heteroatoms are selected fromnitrogen, oxygen, and sulfur, a portion of which, including at least oneheteroatom, form a ring, such as aziridine, pyrrolidine, oxazolidine,3-methoxypyrrolidine, 3-methylpiperazine, and the like.

The term “alkoxy” as used herein refers to alkyl as defined hereincombined with a terminal oxygen, such as methoxy, ethoxy, propoxy,3-methylpentoxy, and the like.

The term “halo” or “halogen” refers to fluoro, chloro, bromo, and iodo.

The term “aryl” as used herein refers to an aromatic mono or polycyclicring of carbon atoms, such as phenyl, naphthyl, and the like.

The term “heteroaryl” as used herein refers to an aromatic mono orpolycyclic ring of carbon atoms and at least one heteroatom selectedfrom nitrogen, oxygen, and sulfur, such as pyridinyl, pyrimidinyl,indolyl, benzoxazolyl, and the like.

The term “substituted aryl” or “substituted heteroaryl” as used hereinrefers to aryl or heteroaryl substituted with one or more substituentsselected, such as halo, hydroxy, amino, alkyl or dialkylamino, alkoxy,alkylsulfonyl, cyano, nitro, and the like.

The term “iminoalkylidenyl” as used herein refers to a divalent radicalcontaining alkylene as defined herein and a nitrogen atom, where theterminal carbon of the alkylene is double-bonded to the nitrogen atom,such as the formulae —(CH)═N—, —(CH₂)₂(CH)═N—, —CH₂C(Me)=N—, and thelike.

The term “amino acid” as used herein refers generally toaminoalkylcarboxylate, where the alkyl radical is optionally substitutedwith alkyl, hydroxy alkyl, sulfhydrylalkyl, aminoalkyl, carboxyalkyl,and the like, including groups corresponding to the naturally occurringamino acids, such as serine, cysteine, methionine, aspartic acid,glutamic acid, and the like.

The term “arylalkyl” refers to aryl as defined herein substituted withan alkylene group, as defined herein, such as benzyl, phenethyl,a-methylbenzyl, and the like.

It should be understood that the above-described terms can be combinedto generate chemically-relevant groups, such as “alkoxyalkyl” referringto methyloxymethyl, ethyloxyethyl, and the like, and “haloalkoxyalkyl”referring to trifluoromethyloxyethyl,1,2-difluoro-2-chloroeth-1-yloxypropyl, and the like.

The term “amino acid derivative” as used herein refers generally toaminoalkylcarboxylate, where the amino radical or the carboxylateradical are each optionally substituted with alkyl, carboxylalkyl,alkylamino, and the like, or optionally protected; and the interveningdivalent alkyl fragment is optionally substituted with alkyl, hydroxyalkyl, sulfhydrylalkyl, aminoalkyl, carboxyalkyl, and the like,including groups corresponding to the side chains found in naturallyoccurring amino acids, such as are found in serine, cysteine,methionine, aspartic acid, glutamic acid, and the like.

The term “peptide” as used herein refers generally to a series of aminoacids and amino acid analogs and derivatives covalently linked one tothe other by amide bonds.

The term “releasable linker” as used herein refers to a linker thatincludes at least one bond that can be broken under physiologicalconditions (e.g., a pH-labile, acid-labile, redox-labile, orenzyme-labile bond). It should be appreciated that such physiologicalconditions resulting in bond breaking include standard chemicalhydrolysis reactions that occur, for example, at physiological pH, or asa result of compartmentalization into a cellular organelle such as anendosome having a lower pH than cytosolic pH.

The term “spacer linker” as used herein refers to an organic moiety thatseparates the active agent or releasable linker from the NK-1receptor-binding moiety.

In cancer treatment, the use of conventional chemotherapeutics may belimited due to their indiscriminate accumulation in both cancer andhealthy cells, thereby resulting in dose-limiting toxicities inuntargeted healthy tissues. One mechanism to overcome such generalaccumulation may be selective ligand-targeted delivery of cytotoxicagents to malignant tissues. In particular, various embodiments providefor uses of NK-1 receptor-targeted ligands to provide therapeutic andimaging agents for use in diagnosis and treatment of cancer.

The various embodiments provide NK-1 receptor-binding agent deliveryconjugates that enable improved mechanisms for targeted delivery ofactive agents to cells expressing NK-1 receptors. In particular, thevarious embodiment compounds may include an NK-1 receptor-binding moiety(NK), at least one linker group (L), and at least one active agent (A)(e.g., a drug, a fluorescent dye, a radioimaging agent, and/or acombination thereof). In the various embodiments, the NK-1receptor-binding moiety and the active agent may be bound to the linkergroup.

The linker groups in the various embodiments may include one or morespacer linkers and releasable linkers, and combinations thereof, in anyorder. The various active agents and linker groups discussed below areprovided as non-limiting examples, for which alternatives are describedin International Published Patent Application No. WO2013/126797, thedisclosure which is incorporated herein by reference in its entirety.

Active Agents

The active agents D of the conjugates of the present invention may, invarious embodiments, be therapeutic agents and/or imaging agents. Theonly limitation on suitable therapeutic agents and imaging agents is therequirement that they have a position on the molecule to which can beconjugated the linker L. or that they can be derivatized to possess sucha position without losing the activity of the active moiety orcompromising the ability of the NK-1 receptor-binding moiety to bind toits receptor with high affinity.

The therapeutic agents described herein function through any of a largenumber of mechanisms of action. Generally, therapeutic agents disruptcellular mechanisms that are important for cell survival and/or cellproliferation and/or cause apoptosis. By way of example only, thetherapeutic agents can be any compound known in the art which iscytotoxic, enhances tumor permeability, inhibits tumor cellproliferation, promotes apoptosis, decreases anti-apoptotic activity intarget cells, is used to treat diseases caused by infectious agents,enhances an endogenous immune response directed to the pathogenic cells,or is useful for treating a disease state caused by any type ofpathogenic cell.

Therapeutic agents suitable for use in accordance with this inventioninclude, without limitation, adrenocorticoids and corticosteroids,alkylating agents, antiandrogens, antiestrogens, androgens, aclamycinand aclamycin derivatives, estrogens, antimetabolites such as cytosinearabinoside, purine analogs, pyrimidine analogs, and methotrexate,busulfan, carboplatin, chlorambucil, cisplatin and other platinumcompounds, taxanes, such as tamoxiphen, taxol, paclitaxel, paclitaxelderivatives, Taxotere®, and the like, maytansines and analogs andderivatives thereof, cyclophosphamide, daunomycin, doxorubicin,rhizoxin, T2 toxin, plant alkaloids, prednisone, hydroxyurea,teniposide, mitomycins, discodermolides, microtubule inhibitors,epothilones, tubulysin (e.g., tubulysin B hydrazide), cyclopropylbenz[e]indolone, seca-cyclopropyl benz[e]indolone, O—Ac-seca-cyclopropylbenz[e]indolone, bleomycin and any other antibiotic, nitrogen mustards,nitrosureas, vincristine, vinblastine, and analogs and derivativethereof such as desacetyl vinblastine monohydrazide, colchicine,colchicine derivatives, allocolchicine, thiocolchicine, trityl cysteine,Halicondrin B, dolastatins such as dolastatin 10, amanitins such asa-amanitin, camptothecin, irinotecan, and other camptothecin derivativesthereof, geldanamycin and geldanamycin derivatives, estramustine,nocodazole, MAP4, colcemid, inflammatory and proinflammatory agents,peptide and peptidomimetic signal transduction inhibitors, and any otherart-recognized drug or toxin. Other drugs that can be used in accordancewith the invention include penicillins, dinitrophenol, fluorescein, CpGoligonucleotides, staurosporine and the kinase inhibitors, Sutent,resiquimod and other Toll-like receptor agonists, cephalosporins,vancomycin, erythromycin, clindamycin, rifampin, chloramphenicol,aminoglycoside antibiotics, gentamicin, amphotericin B, acyclovir,trifluridine, ganciclovir, zidovudine, amantadine, ribavirin, and anyother art-recognized antimicrobial compound.

Specific sub-groups of therapeutic agents include, but are not limitedto, radio-therapeutic agents, immunotherapeutic agents, photodynamictherapy agents and chemo therapeutic agents. The skilled artisan willunderstand that there is a wide variety of radio-therapeutics that willbe suitable for use in the conjugates of the present invention. Suitableexamples include, but are not limited to, ⁹⁰Y, ³¹I, ¹ ⁷ ⁷Lu, ⁶ ⁷ Cu,^(U) ¹ ln, ¹⁸⁶Re, ²¹′At, and {circumflex over ( )}Ra.

The skilled artisan will also understand that there is a wide variety ofchemotherapeutics that will be suitable for use in the conjugates of thepresent invention. Suitable examples include, but are not limited to,tubulysin B hydrazide and desacetyl vinblastine monohydrazide,calicheamycin, auristatin, maytansinoids, and any other cytotoxic agentwith IC50 value below 10 nM.

It should also be appreciated that the ligand can be used to target ananomedicines or nanoparticle, including but not limited to a liposome,a lipoplex, a polyplex, a dendrimer, a polymer, a nanoparticle, or avirus. It should further be recognized that the aforementioned particlesmight serve as carriers for DNA, RNA, siRNA, peptides, proteins, andother biologies.

Imaging agents suitable for use in the conjugates of the inventioninclude, but are not limited to, radio-imaging agents, optical imagingagents, PET imaging agents, MRI contrast agents, CT contrast agents, andFRET imaging agents, and other agents that may be used to detect orvisualize a tumor, cancer or transformed cell, whether in vitro, in vivoand ex vivo.

Applications for conjugates comprising radio-imaging agents include, butare not limited to, diagnosis of disease and or locating metastaticdisease, detecting disease recurrence following surgery, monitoringresponse to therapy, development of a radio-therapeutic conjugate andselecting patients for subsequent CCK2R targeted therapy. Radio-imagingagents include radioactive isotopes, such as a radioactive isotope of ametal, coordinated to a chelating group. Illustrative radioactive metalisotopes include technetium, rhenium, gallium, gadolinium, indium,copper, and the like, including isotopes ^(U) ¹ ln, ^(99m)Tc, ⁶ ⁴ Cu, ⁶⁷ Cu, ⁶⁷Ga, ⁶ ⁸ Ga, and the like, or they may include radionuclides thatare effective in radiotherapy.

Illustratively, the following chelating groups are described that can beused with the radio-imaging agents:

wherein R is independently selected in each instance from, for example,H, alkyl, heteroalkyl, cycloalkyl, heterocyclyl, alkenyl, alkynyl, aryl,heteroaryl, arylalkyl, heteroarylalkyl, and the like, each of which isoptionally substituted, wherein one R includes a heteroatom, such asnitro, oxygen, or sulfur, and is the point of attachment of linker L; Xis oxygen, nitrogen, or sulfur, and X is attached to linker L; and n isan integer from 1 to about 5.

Additional illustrative chelating groups are tripeptide ortetrapeptides, including but not limited to tripeptides having theformula:

wherein R is independently selected in each instance from H, alkyl,heteroalkyl, cycloalkyl, heterocyclyl, alkenyl, alkynyl, aryl,heteroaryl, arylalkyl, heteroaryalkyl, and the like, each of which isoptionally substituted. It is to be understood that one R includes aheteroatom, such as nitro, oxygen, or sulfur, and is the point ofattachment of linker L.

Applications for conjugates comprising optical imaging agents includelocating and resecting large tumor masses, delineation of normal andmalignant tissue, intraoperative detection of sentinel lymph nodes, andfluorescent probe for minimally invasive laparoscopic procedures as analternative to second look surgery. The skilled artisan will alsounderstand that there is a wide variety of optical imaging agents thatwill be suitable for use in the conjugates of the present invention. Theonly limitations on suitable optical imaging agents is the requirementthat they have a position on the molecule to which can be conjugated thelinker L, or that they can be derivatized to possess such a position.Examples include, but are not limited to, Oregon Green fluorescentagents, including but not limited to Oregon Green 488, Oregon Green 514,and the like, AlexaFluor fluorescent agents, including but not limitedto AlexaFluor 488, AlexaFluor 647, and the like, fluorescein, andrelated analogs, BODIPY fluorescent agents, including but not limited toBODIPY F1, BODIPY 505, S0456, and the like, rhodamine fluorescentagents, including but not limited to tetramethylrhodamine, and the like,near infra-red fluorescent agents, including but not limited to DyLight680, DyLight 800, 800CW, LS288, S0456, indocyanine green and the like,Texas Red, phycoerythrin, and others. Illustrative optical imagingagents are shown in the following general structure:

where X is oxygen, nitrogen, or sulfur, and where X is attached tolinker L; Y is ORa, NRa₂, or NRa₃₊; and Y′ is 0, NRa, or NRa₂ ₊ ; whereeach R is independently selected in each instance from H, fluoro,sulfonic acid, sulfonate, and salts thereof, and the like; and Ra ishydrogen or alkyl.

According to another aspect, illustrative optical imaging agents areshown in the following general structure:

where X is oxygen, nitrogen, or sulfur, and where X is attached tolinker L; and each R is independently selected in each instance from H,alkyl, heteroalkyl, and the like; and n is an integer from 0 to about 4.

The skilled artisan will also understand that there is a wide variety ofPET imaging agents and FRET imaging agents that will be suitable for usein the conjugates of the present invention. The only limitations onsuitable PET and FRET imaging agents is the requirement that they have aposition on the molecule to which can be conjugated the linker L, orthat they can be derivatized to possess such a position. Examples of PETimaging agents include, but are not limited to, ¹ ⁸ F, ¹¹C, {circumflexover ( )}Cu, ⁶ ⁵ Cu, and the like. Examples of FRET imaging agentsinclude, but are not limited to, ⁶⁴Eu, ⁶⁵Eu, and the like. Itappreciated that in the case of ¹⁸F and ¹¹C, the imaging isotope may bepresent on any part of the linker, or alternatively may be present on astructure attached to the linker. For example in the case of —F,fluoroaryl groups, such as fluorophenyl, difluorophenyl,fluoronitrophenyl, and the like are described. For example in the caseof ¹¹C, alkyl and alkyl aryl are described. Exemplary optical imagingagents include, but are not limited to, fluorescein (FITC), rhodamine,LS288, S0456, IR800CW, or another near infrared dye.

Linkers

Exemplary linkers may include, but are not limited to, hydrophiliclinkers comprised of charged or polar amino acids, sugars orsugar-containing oligomers, and hydrophilic polymers such aspolyethylene glycol.

In a first embodiment, the linker L is L1, L2 or L3, where L1 isHN-Glu-Arg-Asp-CO, L2 is HN-Glu-PS-Glu-PS-CO, and L3 isHN-Octanoyl-Glu-PS-Glu-PS-CO, in which PS has the following formula:

The linkers used in the production of the embodiment compounds may alsocomprise one or more spacer linkers and/or one or more releasablelinkers, and combinations thereof, in any order. It is appreciated thatspacer linkers may be included when predetermined lengths are selectedfor separating the NK-1 receptor-binding moiety from the active agent.It is also appreciated that in certain configurations, releasablelinkers may be included.

In an example, the linker group may be a releasable linker that includesa cleavable bond connecting two adjacent atoms. Following breakage ofthe cleavable bond, the releasable linker may be broken into two or morefragments. In another example the releasable linker may contain acleavable bond that connects one end of the releasable linker to otherlinkers, to the active agent, or to the NK-1 receptor-binding moiety.Following breakage of the cleavable bond, the releasable linker may beseparated from the other moiety. Examples of a releasable linker mayinclude, but are not limited to, a disulfide group and a carbamategroup.

In a particular application, expression of NK-1 receptors has beenreported in a number of lethal cancers of the brain (glioblastoma,glioma and astrocytoma), skin (melanoma), pancreas, retina(retinoblastoma), nerve (neuroblastoma), larynx, stomach, colon andbreast. Therefore, in various embodiments, the NK-1 receptor-bindingagent delivery conjugates may be used to treat and/or diagnose diseasestates and/or tissues characterized by the presence of a pathogenic cellpopulation having accessible NK-1 receptors that are uniquely expressed,overexpressed, or preferentially expressed by the pathogenic cells(i.e., not present or present at lower concentrations on non-pathogeniccells). Selective elimination, tracing, or imaging of pathogenic cellsmay therefore be mediated by the binding of the NK-1 receptor-bindingmoiety to the NK-1 receptor.

Specifically, over-expression of NK-1 receptors has been reported inlethal cancers of the brain (glioblastoma, glioma and astrocytoma), skin(melanoma), pancreas, retina (retinoblastoma), nerve (neuroblastoma),larynx, stomach, colon and breast, the various embodiments may be usedto develop an NK1R-targeted NIR dye for use in cancer. Specifically,because NK-1 receptor-binding agent delivery conjugates are typicallyunable to cross the blood brain barrier, they may fail to reach the vastmajority of NK-1 receptors present in normal brain tissues. Instead, anNK-1 receptor-binding agent delivery conjugate may selectivelyaccumulate in NK1R-expressing tumor xenografts with very high affinity,allowing targeted delivery of the active agents to NK-1 receptorexpressing tumor cells.

In the various embodiments, the NK-1 receptor-binding moiety may be anyof a number of ligands, such as a small molecule ligand customized to behighly selective for NK-1 receptors. For example, the small moleculeligand may have a dissociation constant (KD) of around 13 nM for theNK-1 receptor.

An example NK-1 receptor-binding ligand (“NK1RL”) may be synthesizedfrom a high affinity NK-1 receptor antagonist(2S,3S)-3-{[3,5-bis(trifluoromethyl)benzyl]oxy}-2-phenylpiperidine(“L733060” and acetic acid (AcOH), and has the formula:

In a first set of embodiments, the active agent of the NK-1receptor-binding agent delivery conjugate may be a drug for therapy inorder to treat NK-1 receptor positive tumor bearing tissue, as may beshown in mouse xenograft models expressing NK-1 receptors. Example drugsmay include, but are not limited to, tubulysin B hydrazide (TubH)) anddisulfide-activated desacetyl vinblastine hydrazide (DAVBH). In suchdrug conjugates, an example linker group may include PEG2 coupled to apeptide that contains a chelating portion of etarfolatide. Inparticular, the linker group may be PEG2-Arg-Asp-Lys-2,3diaminopropionic acid (DAP)-Asp-Cys (referred to herein as “EC20peptide” linker), which has a formula of:

In another example drug conjugate, the linker group may be synthesizedfrom a cysteine-based resin, similar to the EC20 peptide linker, but maycontain PEG2 that is coupled to a shorter peptide compared to the EC20peptide linker. In particular, the linker group may be PEG2-Arg-Asp-Cys(referred to herein as “Cys peptide” linker), which has a formula of:

In another example drug conjugate, the linker group may be synthesizedfrom a cysteine-based resin, similar to the EC20 peptide linker and Cyspeptide linker, but may contain any of a variety of PEG structurescoupled to a shorter peptide that contains the chelating portion ofetarfolide. In particular, the linker group may be PEG,-2,3diaminopropionic acid (DAP)-Asp-Cys (referred to herein as “short EC20”linker). For example, a short EC20 linker may include PEG2 (PEG2-basedshort EC20 linker) and have a formula of:

Another example PEG short EC20 linker may include PEG 12 (PEG12-basedshort EC20 linker) and have a formula of:

Another example PEG short EC20 linker may include PEG36 (PEG36-basedshort EC20 linker) and have a formula of:

In another example, the linker group may contain one or more peptidesugar (PS) chains in addition to the short EC20 linker. For example, thelinker group may include two PS chains coupled to a PEG2-based shortEC20 linker. Such linker group (PS2-PEG2-based short EC20 linker) mayhave a formula of:

In another example, the linker group may include a short EC20 linkerthat is free of PEG (i.e., PEGO-based short EC20 linker), which iscoupled to one or more PS chain. Such linker group (PS2-PEGO-based shortEC20 linker) may have a formula of:

In another set of embodiments, the active agent of the NK-1receptor-binding agent delivery conjugate may be a radioimagingconjugate. For example, the active agent (i.e., radioimaging agent) maybe a radiotagged element for use in single-photon emission computedtomography (SPECT) and/or positron emission tomography (PET) imaging oftumor tissue, and may be shown in mouse xenograft models expressing NK-1receptors.

In some embodiment radioimaging conjugates, the active agent may be aradiopharmaceutical containing a chelator linked to a radiotag, such astechnetium-99m (⁹⁹ ^(m) Tc). “^(m)Tc is a metastable nuclear isomer oftechnetium-99 (⁹⁹Tc), and is the most commonly used medicalradioisotope. When used as a radioactive tracer, ⁹⁹ ^(m) Tc can bedetected in the body by medical equipment (gamma cameras). ⁹⁹ ^(m) Tcemits readily detectable 140 keV gamma rays (these 8.8 pm photons areabout the same wavelength as emitted by conventional X-ray diagnosticequipment) and its half-life for gamma emission is 6.0058 hours (meaning93.7% of it decays to ⁹ ⁹ Tc in 24 hours). The “short” physicalhalf-life of the ⁹⁹ ^(m) Tc isotope and its biological half-life of 1day (in terms of human activity and metabolism) allow for scanningprocedures that collect data rapidly but keep total patient radiationexposure low. The same characteristics make the isotope suitable onlyfor diagnostic but never therapeutic use.

In other radioimaging conjugates, the active agent may include aradionuclide suitable for use as a tracer in PET imaging. For example,Copper-64 (⁶⁴Cu) is a positron emitter that may be well suited for invitro and in vivo characterization of peptide probes. Radioimagingconjugates in which ⁶⁴Cu labeling is used may also be applied to othercopper isotopes and transition metal isotopes for the purposes ofradionuclide imaging. In some embodiments, a radioimaging agent may be a⁶⁴Cu radio-labeled chelator of 1,4,7-triazacyclononane-1,4,7-triaceticacid (NOTA). An example radioactive imaging conjugate may include NK1RLas the NK-1 receptor binding moiety, a linker, and a ⁶ ⁴ Cu-labeled NOTAgroup as the active agent, according to the formula:

Other compounds may be used in the radioimaging agent and may be labeledwith alternative isotopes for PET imaging instead of ⁶ ⁴ Cu, includingbut not limited to, 1 l-indium, 18-fluorine, 68-gallium, etc. Forexample, in another embodiment the radioimaging agent may be an ¹¹¹inradio-labeled chelator of“4,7,10-tetraazacyclododecane-1,4,7,10-triacetic acid (DOTA). Theradioactive imaging conjugate may include NKIRL as the NK-1 receptorbinding moiety, a PEG2 linker, and a ¹“in-labeled DOTA group as theactive agent, according to the formula:

In an example radioimaging conjugate, the NK-1 receptor binding moietymay be NK1RL, the linker group may be a short EC20 linker (e.g.,PEG2-based short EC20 linker), and the active agent may be a ⁹⁹ ^(m) Tcradiotag, linked to the chelating portion of the linker group, accordingto the structure:

Alternatively, the linker group may be a different short EC20 linker(e.g., PEG36-based short EC20 linker), and the active agent may be ⁹^(9m) Tc linked to the chelating portion of the linker group, accordingto the structure:

In another example radioimaging conjugate, the NK-1 receptor bindingmoiety may be NKIRL, the linker group may be PS2 coupled to a short EC20linker (e.g., PEG2-based short EC20 linker), and the active agent may bea radiotag, such as technetium-99m (^(99m)Tc), linked to the chelatingportion of the linker group, according to the structure:

In another set of embodiments, the active agent of the NK-1receptor-binding agent delivery conjugate may be a fluorescent compound(i.e., fluorescent agent) for use in optical imaging andfluorescence-guided surgery of certain tumors, as may be shown in livetumor-bearing mice in which NK-1 receptors are expressed in tumortissue. Although most fluorescent agent operate in visible orultraviolet parts of the spectrum, near infrared (NIR) area may bebetter suited for fluorescence detection and imaging for scenarios inwhich high signal-to-noise ratio is important. Therefore, a fluorescentimaging conjugate may include any of a variety of fluorescent agents.

In various embodiments, in tumor uptake in malignant lesions,specificity of the NK-1 receptor-binding agent delivery conjugates tothe NK-1 receptor may be confirmed using an active agent containing afluorescent agent. In particular, fluorescent visualization of the tumorcells may be performed using a fluorescent imaging conjugate, and NK-1receptor specificity may be established by demonstrating blockade of thefluorescence through administration of excess unlabeled NK-1 receptorbinding moiety (e.g., NKIRL). In vivo and in vitro studies of thevarious embodiment conjugates may be performed using HEK 293 cells whichare transduced with TACR1 to express NK-1 receptors.

In another example fluorescent imaging conjugate, the NK-1 receptorbinding moiety may be NK1RL, the linker group may be a PEG structure(e.g., PEG36), and the fluorescent agent may be an NIR fluorescent dyeS0456, according to the structure:

In another example fluorescent imaging conjugate, the NK-1 receptorbinding moiety may be NKIRL, the linker group may be the EC20 peptidelinker, and the fluorescent agent may be an NIR fluorescent dyeLS288-maleimide, according to the structure:

In alternative embodiment fluorescent imaging conjugates, the NK-1receptor-binding moiety may be a small molecule ligand other than NKIRL.For example, the NK-1 receptor-binding moiety may be a sulfurpentafluoride-containing small molecule (“NK1RL-SF5”) according to oneof the following structures:

In another example fluorescent imaging conjugate, the NK-1receptor-binding moiety may be NK1RL-SF5, and the linker group may bePEG2 coupled to a tyrosine-containing peptide, the group being referredto herein as a “tyrosine peptide linker.” The active agent may be theNIR fluorescent dye S0456, providing an NK-1 receptor-binding moiety mayaccording to the structure:

In another example fluorescent imaging conjugate, the NK-1receptor-binding moiety may be NK1RL-SF5, and the linker group may be aPEG and a peptide group. For example the linker group may be PEG2coupled to a lysine-containing peptide, with the group being referred toherein as a “lys peptide linker.” The active agent may be a rhodaminecompound, providing an NK-1 receptor-binding moiety may according to thestructure:

In particular, rhodamine dyes may be used, for example, as a tracerwithin water to determine the rate and direction of flow and transport.Further, since rhodamines may be detected easily and inexpensively withfluorometers, embodiment fluorescent imaging conjugates in which theactive agent in rhodamine may be used in a variety of biotechnologyapplications (e.g., fluorescence microscopy, flow cytometry,fluorescence correlation spectroscopy, ELISA, etc.) as part of otherprocesses to develop, test, and quantify effectiveness of variousconjugates.

Non-limiting examples of fluorophores suitable for use as an activeagent in other embodiments may include, without limitation,N,N-dimethyl-4-benzofurazansulfonamide (DBD),4-(2-Aminoethylamino)-7-(N,N-dimethylsulfamoyl)benzofurazan (DBD-ED),indocyanine green (ICG), a Dylight-700 such as Dylite-700-2B, IR820;3,3′-Diethylthiatricarbocyanine iodide (DTTCI), LS277, a cypatem, and acoumarin.

In an embodiment, the active agent may be a drug that includes anitrogen atom, and the linker group may include haloalkylenecarbonyl,optionally substituted with a substituent X², where thehaloalkylenecarbonyl may be bonded to the drug nitrogen to form anamide.

In another embodiment, the active agent may be a drug that includes anoxygen atom, and the linker group may include haloalkylenecarbonyl,optionally substituted with a substituent X2, where thehaloalkylenecarbonyl may be bonded to the drug oxygen to form an ester.

In another embodiment, the active agent may be a drug that includes adouble-bonded nitrogen atom, and the linker group may includealkylenecarbonylamino and 1-(alkylenecarbonylamino)succinimid-3-yl,where the linker group may be bonded to the drug nitrogen to form ahydrazone.

The drug can include a double-bonded nitrogen atom, and in thisembodiment, the releasable linkers can be alkylenecarbonylamino and1-(alkylenecarbonylamino)succinimid-3-yl, and the releasable linker canbe bonded to the drug nitrogen to form an hydrazone.

The various embodiments may be understood by reference to the followingnon-limiting examples, which are provided by way of illustration only.

Example 1

General

NK-1 receptor-binding agent delivery conjugates were developed forclinical and diagnostic application. In these processes, moisture andoxygen sensitive reactions were carried out under an argon atmosphere.Solid phase peptide synthesis (SPPS) was performed using a standardpeptide synthesis apparatus (Chemglass, Vineland, N.J.). Columnchromatography was performed with silica gel as the solid phase and TLCwas conducted on silica gel TLC plates and visualized under UV light.All peptides and their conjugates were purified by preparative reversephase (RP)-high performance liquid chromatography (HPLC) and wereanalyzed by analytical RP-HPLC. ¹H and ¹³C NMR spectra were acquiredwith Bruker 400 or 500 MHz NMR spectrophotometer and the signals arerecorded in ppm with reference to residual CHCI3 (7.27 ppm) or DMSO(2.50 ppm) and data are reported as s=singlet, d=doublet, t=triplet,q=quartet, m=multiplet, b=broad with coupling constants in Hz.

Electrospray ionization-high resolution mass spectrometry (ESI-HRMS) wasperformed utilizing the appropriate polypropylene glycol standards.Radioactivity was counted on a Packard γ-counter (Packard InstrumentCompany, Meriden, Conn.). The tumor imaging was performed using a KodakImage Station (In-Vivo FX, Eastman Kodak Company, New Haven, Conn.).

HEK 293 cell lines stably transfected with neurokinin-I receptor (NK1R)were utilized. Cell lines were cultured in Dulbecco's modified Eagle'smedium (GIBCO) supplemented with 10% fetal bovine serum, G4 18 disulfate(Sigma Aldrich 400 μg/mL), and 1% penicillin streptomycin at 37° C. in ahumidified 95% air 5% C0₂atmosphere.

Athymic male nu/nu mice were purchased from Harlan Laboratories(Indianapolis, Ind.), maintained on normal rodent chow and housed in asterile environment on a standard 12h light and dark cycle for theduration of the study. All animal procedures were approved by the PurdueAnimal Care and Use Committee (PACUC) in accordance with NIH guidelines.

Synthesis of NK1RL

The NK-1 receptor-binding moiety was an NK-1 receptor ligand (NK1RL)formed from the starting compound(2S,3S)-3-((3,5-bis(trifluoromethyl)benzyl)oxy)-2-phenylpiperidine(L-733, 060), a high affinity NK-1 receptor antagonist. The L-733,060compound was formed according to the literature procedure. Formation ofNK1RL from L-733,060 was performed according to the following steps,illustrated in FIG. A.

To the(2S,3S)-3-((3,5-bis(trifluoromethyl)benzyl)oxy)-2-phenylpiperidine (1,0.065 g, 0.16 mmol) in dry THF (1.5 mL), were added tri ethylamine(0.056 mL, 0.4 mmol, 2.5 equiv) followed by tert-butyl-2-bromo acetate(0.035 mL, 0.24 mmol, 1.5 equiv) under N2. The reaction was stirred for16 hours at room temperature. The reaction was quenched with water and2% HCl solution and extracted with EtOAc (3×5 mL). The combined organiclayers were washed with brine, dried (Na₂S0₄), filtered and concentratedand residue was purified by silica-gel column chromatography(hexance:EtOAc, 4:1) to give product, 2 (0.075 g, 92%).

To the ester (0.075 g, 0.14 mmol) in dry CH₂Cl₂ was addedtrifluoroacetic acid (TFA) (20 equiv) and stirred for 4 hours at roomtemperature. The excess of TFA was removed and diluted with water,extracted with CH2Cl2 (3×5 mL). The combined organic layers were washedwith brine, dried (Na₂S0₄) and concentrated. The residue obtained waspurified by flash silica-gel column chromatography (hexane:EtOAc, 3:7)to give acid, 3 (0.06 g, 90/6) as a white solid.

Synthesis of NK1RL-EC20 Peptide Linker

An EC20 peptide linker was created from PEG2 and an amino acid chain(Arg-Asp-Lys-DAP-Asp-Cys). A compound of the linker and NK1receptor-binding moiety NK1RL (NK1RL-EC20 peptide linker) wassynthesized through solid phase peptide synthesis (SPPS) and purified.Synthesis and purification of the NK1RL-EC20 peptide linker compound wasperformed according to the following steps.

H-Cys(4-methoxytrityl)-Wang resin (150 mg, 0.64 mmol) was swollen indichloromethane (2×5 mL) and dimethylformamide (DMF) (2×3 mL) whilebubbling under argon. After swelling the resin in DMF, a solution offluorenylmethyloxycarbonyl chloride-aspartic acid-4-tert-butyl ester(Fmoc-Asp(OtBu)-OH) (2.5 equiv),benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate(PyBOP) (2.5 equiv), and N,N-diisopropylethylamine (DIPEA) (5 equiv) inDMF were added. The resulting solution was bubbled under argon for 4hours and drained, and the resin was washed with DMF (3×5 mL) andisopropyl alcohol (i-PrOH) (3×5 mL). Fmoc deprotection was carried outusing 20% piperidine in DMF (3×10 mL) and the resin was washed with DMF(3×5 mL) and i-PrOH (3×5 mL). Ninhydrin tests as described in Kaiser etal. “Color test for detection of free terminal amino groups in thesolid-phase synthesis of peptides,” Anal Biochem. 1970 April;34(2):595-8 (“Kaiser tests”) were conducted to assess coupling anddeprotection steps. The above sequence was repeated for 5 more couplingsteps. Final coupling was done by L-733,060-AcOH (3, 1.5 equiv) undersame conditions for 12 hours.

The resin was washed with DMF (3×5 mL) and i-PrOH (3×5 mL) and allowedto dry under nitrogen. The peptide linker was then cleaved from theresin using a mixture of trifluoroacetic acid(TFA):3/40:triisopropylsilane:ethanedithiol cocktail (92.5:2.5:2.5:2.5).The solution was bubbled twice under nitrogen for 15 min, drained,concentrated, and then precipitated by addition of cold diethyl ether.Crude product was collected by centrifugation, washed three more timeswith cold diethyl ether, dried under vacuum, and then purified bypreparative reverse-phase HPLC (Waters, XBridge™ Prep C18, 5 μm; 19×100mm column, mobile phase A=20 mM ammonium acetate buffer, pH 5,B=acetonitrile, gradient 10-100% B in 30 min, 13 mL/min, λ=280 nm). Purefractions were analyzed by liquid chromatography mass spectrometry(LC-MS) and low resolution mass spectrometry (LR-MS) and were pooled andlyophilized to furnish a compound of NK1RL-EC20 peptide linker(yield=63.80 mg, 50%. LR-MS (m/z): 1323.53 M+H)+. UV/vis: Xmax=254 nm).The ligand-linker compound formed from NK1RL and the EC20 peptide linkeris shown in the schematic equation below:

Synthesis of NKIRL-Cys Peptide Linker

A Cys peptide linker was created from PEG2 and an amino acid chain(Arg-Asp-Cys). The ligand-linker compound of NKIRL-Cys peptide linkerwas synthesized through SPPS with fewer amino acids starting fromCysteine resin and purified. Synthesis and purification of NKIRL-Cys wasperformed using substantially the same procedure as for NK1RL-EC20peptide linker.

Synthesis of Drug Conjugates

NK1RL-EC20peptide Unker-TubH

To a solution of NK1RL-EC20 peptide linker (2.80 mg, 2.1 15 umol) in dryDMSO (0.1 mL) at 0° C., disulfide-activated-TubH (2.0 mg, 2.115 μmol)followed by DIPEA (3 μL, 0.21 15 μmol) were added. The reaction mixturewas stirred for 3 hours at room temperature, and the crude product waspurified by RP-HPLC (Waters, XBridge™ Prep C18, 5 pam; 19×100 mm column,mobile phase A=20 mM ammonium acetate buffer, pH 7, B=acetonitrile,gradient 10-100% B in 30 min, 13 mL/min,)=280 nm). Pure fractions wereanalyzed by LC-MS and LR-MS and were pooled and lyophilized to affordNK1RL-EC20 peptide linker-TubH. Yield=3.26 mg, 68%. LR-MS (m/z): 2270.53(M+H)+. UV/vis: Xmax=254 nm.

A schematic equation showing the formation of NK1RL-EC20 peptidelinker-TubH is shown below:

NKIRL-Cys Peptide Linker-TubH

Further, the same procedural steps were followed using NKIRL-Cys peptidelinker discussed above in order to synthesize an NKIRL-Cys peptidelinker-TubH compound which was then and purified through RP-HPLCaccordingly as described above and characterized.

A schematic equation showing the formation of NKIRL-Cys peptidelinker-TubH is shown below:

NK1RL-EC20peptide linker-DAVBH

To a solution of NK1RL-EC20 linker (1.30 mg, 0.9819 μmol) in dry DMSO(0.1 mL) at 0° C., disulfide-activated-DAVBH (1.16 mg, 1.1782 μmol)followed by DIPEA (2.5 uL, 19.637 umol) were added. The reaction mixturewas stirred for 4 hours at room temperature, and the crude product waspurified by RP-HPLC (Waters, XBridge™ Prep C18, 5 μm; 19×100 mm column,mobile phase A=20 mM ammonium acetate buffer, pH 7, B=acetonitrile,gradient 10-100% B in 30 min, 13 mL/min, λ=280 run). Pure fractions wereanalyzed by LC-MS and LR-MS and were pooled and lyophilized to affordNK1RL-EC20 peptide linker-DAVBH (yield=1.40 mg, 65%. LR-MS (m/z):2195.43 (M+H)+. UV/vis: λmax=254 nm).

A schematic equation showing the formation of NK1RL-EC20 peptidelinker-DAVBH is shown below:

NKIRL-Cys Peptide Linker-DAVBH

Further, the same procedural steps were followed using NKIRL-Cys peptidelinker discussed above in order to synthesize an NKIRL-Cys peptidelinker-DAVBH conjugate which was then and purified through RP-HPLCaccordingly as described above and characterized.

A schematic equation showing the formation of NKIRL-Cys peptidelinker-DAVBH is shown below:

Cytotoxicity Study (1050) for Drug Conjugates

HEK 293 NK1R cells (50,000 cells/well) were seeded into 24 well plates(BD Purecoat Amine, BD Biosciences) and allowed to grow to formmonolayer over 24 to 48 hours. The old medium was replaced with freshmedium (0.5 mL) containing increasing concentrations of drug conjugates(either targeted or non-targeted NK1RL, and free-ligand) and cells wereincubated for an additional 2 hours at 37° C. Cells were washed (3×0.5mL) with fresh medium and incubated in fresh medium (0.5 mL) for another66 hours at 37° C. The spent medium in each well was replaced with freshmedium (0.5 mL) containing [3H]-thymidine (1 mCi/mL), and the cells wereincubated for additional 4 hours at 37° C. to allow [3H]-thymidineincorporation. The cells were then washed with medium (2×0.5 mL) andtreated with 5% trichloroacetic acid (0.5 mL) for 10 min at roomtemperature. The trichloroacetic acid was replaced with 0.25 N NaOH (0.5mL), cells were transferred to individual scintillation vials containingEcolume scintillation cocktail (3.0 mL), mixed well to form homogeneousliquid and counted in a liquid scintillation analyzer. ICso values werecalculated by plotting % [3H]-thymidine incorporation versus logconcentration of drugs (targeted and non-targeted) using in GraphPadPrism 4.

In vivo Studies for Drug Conjugates

Four- to six-week old male nu/nu mice were maintained on a standard 12hours light-dark cycle and fed on normal mouse chow for the duration ofthe experiment and were inoculated subcutaneously on their shoulderswith HEK 293-NK1R cells (5.0×106 cells/mouse in 50% HC Matrigel) using a25-gauge needle. Growth of the tumors was measured in two perpendiculardirections every 2 days using a caliper, and the volumes of the tumorswere calculated as 0.5×L×W2 (L=measurement of longest axis, and W=axisperpendicular to L in millimeters). Experiments on live mice involved atleast five mice per group and animals were treated with therapeutic drugconjugates (1.6 μmol/kg of body weight) in saline (100 μL) for threeweeks, 3 doses per week (M/W/F), when the tumors reached 75-130 mm³volume (˜3 weeks). Tumor volumes and body weights were also measured ateach dose. In vivo efficacy was evaluated by plotting tumor volumeversus days and % of weight loss/gain versus days on therapy. Threeweeks after treatment, the animals were dissected and selected tissueswere preserved in formalin for histopathology studies. H&E stainedslides for microscopic evaluation were prepared from submitted fixedtissues.

FIG. 1B provides in vivo mice therapeutic data for HEK 293-NK1R tumorxenograft models showing behavior of tumor volumes from the NK1R-EC20peptide linker-TubH conjugate. FIG. 1C provides in vivo mice therapeuticdata on HEK 293-NK1R tumor xenografts model showing behavior bodyweights during the therapy shown in FIG. 1B.

Example 2

As illustrated in FIG. 2, synthesis of NKIRL-Lys peptidelinker-rhodamine conjugate was carried out by synthesizing the NKIRL-Lyspeptide linker, to which NHS-rhodamine was added. Specifically,NKIRL-Lys peptide linker-rhodamine conjugate synthesis was performedusing the following steps.

Synthesis of NKIRL-Lys Peptide Linker

H-Lys (Boc)-2-Cl-Trt resin (80 mg, 0.75 mmol) was swollen indichloromethane (DCM) (2×5 mL) and DMF (2×3 mL) while bubbling underargon. A solution of Fmoc-Asp(OtBu)-OH (2.5 equiv), PyBOP (2.5 equiv),and DIPEA (5 equiv) in DMF was added. The resulting solution was bubbledunder argon for 3 hours and drained, and the resin was washed with DMF(3×5 mL) and i-PrOH (3×5 mL). Fmoc deprotection was carried out using20% piperidine in DMF (3×5 mL). The deprotection solution was removed,and the resin was washed again with DMF (3×5 mL) and i-PrOH (3×5 mL).Kaiser tests were conducted to assess coupling and deprotection steps.The same procedure were followed forFmoc-arginine-(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-sulfonyl)ester (Fmoc-Arg(pbf)-OH), Fmoc-PEG2-CO 2H, and NKIL (L-733,060-aceticacid) couplings, with the reaction being bubbled over night for NK1L.The resin was washed with DMF (3×5 mL) and i-PrOH (3×5 mL) and allowedto dry under nitrogen. The NKIL-peptide linker was then cleaved from theresin using a mixture of 95% trifluoroacetic acid (TFA), 2.5% H₂0 and2.5% triisopropylsilane (TIPS). The solution was bubbled three timesunder nitrogen for 15 min, drained, concentrated, and then precipitatedby addition of cold diethyl ether. Crude product was collected bycentrifogation, washed three more times with cold diethyl ether, driedunder vacuum, and then purified by preparative reverse-phase HPLC(Waters, XBridge™ Prep C18, 5 prn; 19×100 mm column, mobile phase A=20mM ammonium acetate buffer, pH 7, B=acetonitrile, gradient 0-80% B in 30min, 13 mL/min, λ=254 nm). Pure fractions were analyzed by LC-MS(XBridge™ RP18, 3.5 μm; 3.0×50 mm column) and low resolutionelectrospray ionisation mass spectrometry (LR-ESIMS), and were pooledand lyophilized to finish NKIRL-Lys peptide linker (Compound 1 in FIG.2).

Synthesis of NK1RL-Lys Peptide Linker-Rhodamine Conjugate

The purified NK1RL-Lys peptide linker (Compound 1 in FIG. 2) was coupledwith NHS-rhodamine by stirring 1:1.2 ratios of Compound I toNHS-rhodamine in dry DMSO. DIPEA under argon for 12 hours at roomtemperature. The resulting material was purified using RP-HPLC (mobilephase A=20 mM ammonium acetate buffer, pH 7, B=acetonitrile, gradient0-80% B in 30 min, 13 mL/min, λ=280 nm). Pure fractions were combined,concentrated under vacuum, and lyophilized to yield the product,NK1RL-Lys peptide linker-rhodamine (Compound 2 in FIG. 2). The NK1RL-Lyspeptide linker-rhodamine conjugate is a reddish solid, was analyzed byLC-MS and LR-ESIMS.

Synthesis of NK1RL-EC20 Peptide Linker-LS288 Conjugate

NK1RL was synthesized as the NK-1 receptor-binding moiety as discussedabove. Synthesis of NK1RL-EC20 peptide linker-LS288 conjugate isillustrated in FIG. 3, and was carried out using the following steps.

To the NK1RL-EC20 peptide linker (Linker 7) in dry DMSO were addedLS288-maleimide (Compound 8) followed by DIPEA under argon atmosphere atroom temperature. The reaction mixture was stirred for overnight at roomtemperature. The product was precipitated by addition of isopropanol andcollected by centrifugation. The crude product was purified bypreparative reverse phase HPLC using a mobile phase of A=20 mM ammoniumacetate buffer, pH 7; B=acetonitrile; gradient 0-50% B in 30 min, 13mL/min, λ=280 nm. Pure fractions were analyzed by LC-MS and LR-MS andwere pooled and lyophilized to furnish NK1RL-EC20 peptide linker-LS288conjugate (Conjugate 9).

Fluorescent Confocal Microscopy Imaging

HEK 293-NK1R cells (50,000 cells/well in 0.5 mL) were seeded intoconfocal microwell plate (Lab-Tek, Chambered #1.0 BorosilicateCoverglass) and allowed cells to form monolayers over 24 hours. Spentmedium was replaced with fresh medium containing NK1RL-Lys peptidelinker-rhodamine (25 nM) in the presence or absence of 100-fold excessfree ligand and cells were incubated for 1 hour at 37° C. After washingwith fresh medium (3×0.5 mL), confocal images were acquired using aconfocal microscopy (FV 1000, Olympus).

FIG. 4 shows resulting images from the binding studies of the NK1RL-Lyspeptide linker-rhodamine conjugate to HEK 293-NK1R cells. Specifically,the images show incubation of cells for 1 hour at 37° C. in the absence(a) and presence (d) of 100-fold excess of competing agent (NK1RL alone)to the conjugate at 25 nM concentration. The images in (b) are 3×magnification from (a), and in (c) are 3× magnification of white light.

Flow Cytometric Analysis

Procedure

HEK 293 NK1R cells were seeded into a T75 flask and allowed to form amonolayer over 48 hours. After trypsin digestion, released cells weretransferred into centrifuge tubes (1×105 cells/tube) and centrifuged.The medium was replaced with fresh medium containing NKIRL-Rhod (25 nM)in the presence or absence of 100-fold excess unlabeled NK1RL ligand andincubated for 1h at 37° C. After rinsing with fresh medium (3×0.5 mL),cells were re-suspended in PBS (0.5 mL) and cell bound fluorescence wasanalyzed (40,000 cells/sample) using a flow cytometer. Untreated HEK293-NK1R cells in PBS served as a negative control.

Determination if Binding Affinity and Specificity

HEK 293 NK1R cells (50,000 cells/well) were seeded into 24 well plates(BD Purecoat Amine, BD Biosciences) and allowed to grow to confluenceover 48-72 hours. Spent medium in each well was replaced with 0.5 mL offresh medium containing 0.5% bovine serum albumin and increasingconcentrations of the NIR dye conjugates in the presence or absence of100-fold excess of competing ligand (i.e., L-733,060). After incubationfor 1 hour at 37° C. cells were rinsed with incubation solution (2×0.5mL) to remove unbound fluorescence and dissolved in 0.5 mL of 1% aqueoussodium dodecyl sulfate (SDS). Cell associated fluorescence was thendetermined by measuring maximum emission of the resulting solution bytransfer to a quartz cuvette upon excitation of each dye(rhodamine/LS288) at 545/755 ran using an Agilent Technologies CaryEclipse fluorescence spectrophotometer. Experiments were performed intriplicate. The conjugate's dissociation constant (KD) was calculatedfrom a lot of cell bound fluorescence emission (a.u.) versus theconcentration of targeted NIR probe added using the GraphPad Prism 4program and assuming a non-cooperative single site binding equilibrium.

FIG. 5 shows the binding of NKIRL-Lys peptide linker-rhodamine to HEK293-NK1R cells by flow cytometry. FIG. 6A shows the binding affinity ofthe NK1RL-Lys peptide linker-rhodamine conjugate in cultured HEK293-NK1R cells expressing NK-1 receptor. FIG. 6B shows the bindingaffinity of the NK1RL-EC20 peptide linker-LS288-maleimide conjugate.

In Vivo Assays

Implantation if Subcutaneous Tumors Using HEK293-NK1R Cells

Six week old male athymic nu/nu mice (Harlan Laboratories, IN) wereinoculated subcutaneously on their shoulders with HEK 293 NK1R cells(5.0×106 cells/mouse in 50% HC Matrigel) using a 25-gauge needle. Growthof the tumors was measured in two perpendicular directions every 2 daysusing a caliper, and the volumes of the tumors were calculated as0.5×L*W2 (L=measurement of longest axis, and W=measurement of axisperpendicular to L in millimeters). Animals were imaged when the tumorsreached 300-500 mm³ volume (˜2-3 weeks). Experiments on live miceinvolved at least four mice per group. Imaging was then performed asdescribed below.

Fluorescence Imaging and Analysis of Mice

Tumor bearing mice were treated via tail vein (i.v) injection with 10nmol of dye conjugate with 100 fold excess competition [three groups(one is dye, two is competition and three is -ve control (KB tumor)groups), 4 mice/group] and imaged 2 hours post injection using a CaliperIVIS Lumina II Imaging station coupled to ISOON5 160 Andor Nikon cameraequipped with Living Image Software Version 4.0. The 2 hour time pointfor imaging was chosen based on data from previous experiments showingthat a radio labeled conjugate of NK1 yielded the highesttumor-to-background ratio at this time point. The settings were asfollows: lamp level, high; excitation, 745 nM: emission, ICG; epiillumination, binning (M) 4; FOV, 12.5: f-stop, 4; acquisition time, 1second. After completion of whole body imaging, animals were dissectedand selected organs were collected and imaged again for completebiodistribution. All organs were preserved in 25 mL of formalin inpreparation for submission to the Purdue Histology & PhenotypingLaboratory for hematoxylin and eosin staining.

FIG. 6C shows (a-b) HEK 293-NK1R tumor xenograft model mice treated withconjugates in which LS288 is the active agent, (c-d) blocking images forthe HEK 293-NK1R tumor xenograft model mice with the treatment in a-b,and (e-f) NK1R-negative tumor xenograft model mice treated withconjugates in which LS288 is the active agent. FIG. 6D is abiodistribution study of the imaged mice in FIG. 6C.

Example 3

Synthesis of the NK1RL-EC20 Peptide Linker-“^(m)Fc Conjugate

A solution of sodium pertechnetate (1.0 mL, 15 mCi) was added to a vialcontaining a lyophilized mixture of NKIRL-EC20 peptide linker (0.178mg), sodium a-D-glucoheptanoate dehydrate (80 mg), stannous chloridedihydrate (0.8 mg), and sufficient NaOH to achieve pH of 7.2 uponrehydration with water. After adding sodium pertechnetate (15 mCi), thevial was heated in a boiling water bath for 18 minutes and then cooledto room temperature before use. The labeling efficiency, radiochemicalpurity, and radiochemical stability were analyzed by RP-HPLC.

In Vitro Studies

HEK 293 NK1R cells (50,000 cells/well) were seeded into 24 well plates(BD Purecoat Amine, BD Biosciences) and allowed to grow to confluenceover 48-72 hours. Spent medium in each well was replaced with 0.5 mL offresh medium containing 0.5% bovine serum albumin and increasingconcentrations of NKIRL-EC20 peptide linker-⁹⁹ ^(m) Tc in the presenceor absence of 100-fold excess of competing ligand, i.e., L-733,060.After incubation for 1 hour at 37° C. cells were rinsed with incubationsolution (3×0.5 mL) to remove unbound radioactive material and cellswere dissolved in 0.25 M NaOH aqueous (0.5 mL) solution. The dissolvedcells were transferred into individual γ-counter tubes and radioactivitywas counted using a γ-counter. The binding constant (Kd) was calculatedby plotting bound radioactivity versus the concentration of targetedradiotracer using GraphPad Prism 4 program, illustrated in FIG. 7A.

In Vivo Studies

Four- to six-week old male nu/nu mice were inoculated subcutaneously ontheir shoulders with HEK 293 NK1R cells (5.0×106 cells/mouse in 50% HCMatrigel) using a 25-gauge needle. Growth of the tumors was measured intwo perpendicular directions every 2 days using a caliper, and thevolumes of the tumors were calculated as 0.5×L*W2 (L=measurement oflongest axis, and W=axis perpendicular to L in millimeters). Animalswere treated with NK1RL-EC20 peptide linker-⁹⁹ ^(m) Tc (1.34 nmol, 150μCi) in saline (100 μL) when the tumors reached 300-500 mm3 volume (˜3weeks). Experiments on live mice involved at least four mice per group,animals were sacrificed by C0₂ asphyxiation at different time points asdescribed below. Images were acquired by a Kodak Imaging Station incombination with CCD camera and Kodak molecular imaging software(version 4.0) (radioimages: illumination source=radio isotope,acquisition time=2 and 4 min, f-stop=0, focal plane=5, FOV=162.5,binning=4; White light images: illumination source=white lighttransillumination, acquisition time=0.175 seconds, f-stop=11, focalplane=5, FOV=162.5 with no binning).

Following imaging, animals were dissected and selected tissues werecollected into pre-weighed γ-counter tubes. Radioactivity of pre-weighedtissues and NK1RL-EC20 peptide linker-⁹⁹ ^(m) Tc (1.34 nmol, 150 μCi) insaline (100 μ) was counted in a γ-counter. CPM values were decaycorrected and results were calculated as % ID/gram of wet tissue andtumor-to-tissue ratios.

FIG. 7B is a set of whole body mice images of mice for NK1RL-EC20peptide linker-99mTc conjugate, showing on the left a HEK 293-NK1R tumorxenograft model treated with NK1RL-EC20 peptide linker-⁹⁹ ^(m) Tcconjugate, and on the right the HEK 293-NK1R tumor xenograft model agedmouse with blocking.

FIG. 7C shows results from a biodistribution study of mice images forthe NK1RL-EC20 peptide linker-⁹⁹ ^(m) Tc conjugate. For each area, theleft-most bar corresponds to administration of NK1RL-EC20 peptidelinker-⁹⁹ ^(m) Tc conjugate, and the right-most bar shows a competitiveNK-1 receptor ligand labeled with ″^(m)Tc. FIG. 7D is a set of wholebody mice images on SPECT-CT for NK1 RL-EC20 peptide linker-99mTcconjugate in HEK 293-NK1R tumor xenograft model mice. FIG. 7E is a setof whole body mice images on SPECT-CT for NK1RL-EC20 peptidelinker-^(99m)Tc conjugate in NK1R-negative tumor xenograft model mice.

Example 4

Synthesis of the NK1RL-PEG2-NOTA-Conjugate

NOTA-NHS ester (11 mg, 0.0016 mmol), followed by DIPEA amine in dry DMSOunder argon were added to a purified NK1RL-PEG2 linker (10 mg, 0.0016mmol), created according to procedures described above. The reactionmixture was stirred for 12 hours at room temperature. The reactionprogress was confirmed by LC-MS and purified by RP-HPLC (mobile phaseA=20 raM ammonium acetate buffer, pH 7, B=acetonitrile, gradient 10-100%B in 30 minutes, 13 mL/min, λ=254 nm). Pure fractions were combined,concentrated under vacuum, and lyophilized to yield the NK1 RL-PEG2-NOTAconjugate, which was analyzed by LC-MS and LR-ESIMS.

In Vivo Studies

A NK-1 receptor-binding radionuclide delivery conjugate was preparedthat contained NK1RL, a PEG2 linker, and {circumflex over ( )}Cu-labeledchelator (radiochemical purity: 99%) with Specific Activity (SA) of 3.7MBq{circumflex over ( )}g. Four NK1R-transduced xenografts (HEK293-NK1R) were prepared, as well as four non-transduced xenografts (HEK293-WT) using athymic nude mice. Doses of 140-160 uCi (˜2 nmoles) ofligand per mouse were intravenously administered, and the models wereimaged at 1, 4 and 20 hours post-injection using PET. The images wereanalyzed in a region of interest (ROI) around the tumor xenograftactivity, and percentage injected dose per mL (% ID/mL) values werecalculated from the mean activity in the ROIs. FIG. 8A is a plot showingROI activity for the NK1R-transduced and non-transduced xenografts.Therefore, the data showed that the NK1RL-PEG2-NOTA-⁶ ⁴ Cu conjugatespecifically accumulated in NK1R-transduced xenografts, but not innon-transduced murine model xenografts. FIG. 8B is a plot showing the{circumflex over ( )}Cu-NK1R ligand uptake ratio between theNK1R-transduced and non-transduced xenografts in various areas at 20hours post-injection.

Analysis of these data showed that that the ratio betweenNK1R-transduced and non-transduced xenografts was highest at 20 hourspost-injection, which may indicate that this radioactive imagingconjugate is more suitable for imaging at a relatively late time pointafter administration.

FIG. 8C is a set of HEK 293-NK1R tumor xenograft model mice on PET usinga conjugate in which the active agent has ⁶⁴Cu.

Example 5

Synthesis of the NK1RL-PEG36-Based Short EC20 Linker-{circumflex over( )}Tc Conjugate

A solution of sodium pertechnetate (1.0 mL, 15 mCi) was added to a vialcontaining a lyophilized mixture of NK1RL-EC20 peptide linker (0.178mg), sodium a-D-glucoheptanoate dehydrate (80 mg), stannous chloridedihydrate (0.8 mg), and sufficient NaOH to achieve pH of 7.2 uponrehydration with water. After adding sodium pertechnetate (15 mCi), thevial was heated in a boiling water bath for 18 minutes and then cooledto room temperature before use. The labeling efficiency, radiochemicalpurity, and radiochemical stability were analyzed by RP-HPLC.

In Vitro Studies

HEK 293 NK1R cells (50,000 cells/well) were seeded into 24 well plates(BD Purecoat Amine, BD Biosciences) and allowed to grow to confluenceover 48-72 hours. Spent medium in each well was replaced with 0.5 mL offresh medium containing 0.5% bovine serum albumin and increasingconcentrations of NKIRL-PEG36-containing short EC20 linker-^(99m)Tc inthe presence or absence of 100-fold excess of competing ligand, i.e.,L-733,060. After incubation for 1 hour at 37° C., cells were rinsed withincubation solution (3×0.5 mL) to remove unbound radioactive materialand cells were dissolved in 0.25 M NaOH aqueous (0.5 mL) solution. Thedissolved cells were transferred into individual γ-counter tubes andradioactivity was counted using a γ-counter. The binding constant (Kd)was calculated by plotting bound radioactivity versus the concentrationof targeted radiotracer using GraphPad Prism 4 program. FIG. 9Aillustrates this binding data for NK1RL-PEG36-containing short EC20linker-⁹⁹ ^(m) Tc in the presence of the competing ligand (top) andwithout the competing ligand (bottom).

In Vivo Studies

Four- to six-week old male nu/nu mice were inoculated subcutaneously ontheir shoulders with HEK 293 NK1R cells (5.0*106 cells/mouse in 50% HCMatrigel) using a 25-gauge needle. Growth of the tumors was measured intwo perpendicular directions every 2 days using a caliper, and thevolumes of the tumors were calculated as 0.5×L*W2 (L=measurement oflongest axis, and W=axis perpendicular to L in millimeters). Animalswere treated with NK1RL-PEG36-containing short EC20 linker-⁹⁹ ^(m) Tc (2nmol, 150 μCi) in saline (100 μL) when the tumors reached 300-500 mm³volume (˜3 weeks). Experiments on live mice involved three mice pergroup, animals were sacrificed by C0₂ asphyxiation at different timepoints as described below. Images were acquired by a Kodak ImagingStation in combination with CCD camera and Kodak molecular imagingsoftware (version 4.0) (radioimages: illumination source=radio isotope,acquisition time=2 and 4 min, f-stop=0, focal plane=5, FOV=162.5,binning=4; White light images: illumination source=white lighttransillumination, acquisition time=0.175 sec, f-stop=11, focal plane=5,FOV=162.5 with no binning).

Following imaging, animals were dissected and selected tissues werecollected into pre-weighed γ-counter tubes. Radioactivity of pre-weighedtissues and NK1RL-PEG36-containing short EC20 linker-⁹⁹ ^(m) Tc (2 nmol,150 μCi) in saline (100 μL) was counted in a γ-counter. CPM values weredecay corrected and results were calculated as % ID/gram of wet tissueand tumor-to-tissue ratios.

FIG. 9B is a set of whole body mice images of mice forNK1RL-PEG36-containing short EC20 linker-⁹⁹ ^(m) Tc conjugate, showingin the upper row a HEK 293-NK1R tumor xenograft model group with a leadshield positioned to allow observation of the relative radioactivity inonly the tumor areas. The lower row shows a HEK 293-NK1R tumor xenograftmodel group treated with NK1RL-PEG36-containing short EC20linker-^(99m)Tc conjugate without shielding.

FIGS. 9C and 9D show results from a biodistribution study of mice imagesfor the NKIRL-PEG36-containing short EC20 linker-″^(m)Tc at 2 hours and8 hours post-injection, respectively. For each area, the left-most barcorresponds to administration of NK1RL-PEG36-containing short EC20linker-″^(m)Tc conjugate, and the right-most bar shows a competitiveNK-1 receptor-binding ligand (L-733,060) labeled with ^(99m)Tc.

Example 6

As illustrated in FIG. 10A, synthesis of NKIRL-SF5-Lys peptidelinker-rhodamine conjugate was carried out by synthesizing the NK1RL-SF5NK-1 receptor-binding moiety, which was used to create the NK1RL-SF5-Lyspeptide linker, to which NHS-rhodamine was added. Specifically,NK1RL-SF5-Lys peptide linker-rhodamine conjugate synthesis was performedusing the following steps.

Synthesis of NK1RL-SF5

Synthesis of an alternative NK-1 receptor-binding moiety NK1RL-SF5,discussed above, was carried out using the following steps.

Starting with(2S,3S)-3-(3-pentatrifluoromethyl)benzyloxy)-2-phenylpiperidine (1,0.055 g, 0.00013 mmol) in dry THF (1.0 mL), were added tri ethylamine(TEA) (0.048 mL, 0.00034 mmol, 2.5 equiv), followed bytert-butyl-2-bromo acetate (0.03 mL, 0.00021 mmol, 1.5 equiv) under N₂.The reaction was stirred for 16 hours at room temperature, The reactionwas quenched with water and 2% HCl solution, and extracted with ethylacetate (EtOAc) (3×5 mL). The combined organic layers were washed withbrine, dried with sodium sulfate (Na₂S0₄), filtered, and concentrated.The resulting residue was purified using silica-gel columnchromatography (hexance:EtOAc, 4:1) to give an intermediary ester (0.070g, 93%) according to the formula:

TFA (20 equiv) was added to the intermediary ester (0.07 g, 0.00013mmol) in dry CH2C2, and stirred for 4 hours at room temperature. Theexcess of TFA was removed and diluted with water, extracted with CH2C12(3×5 mL). The combined organic layers were washed with brine, dried(Na₂SO₄) and concentrated. The residue obtained was purified by flashsilica-gel column chromatography (hexane:EtOAc, 3:7) to give a productNK1RL-SF5 (0.052 g, 90%).

Synthesis of NK1RL-SFS-Lys Peptide Linker

An NK1RL-SF5-Lys peptide linker was synthesized by following ananalogous procedure to that used to synthesize the NK1RL-Lys peptidelinker as discussed above with respect to FIG. 2. The NK1RL-SF5-Lyspeptide linker was then purified through RP-HPLC and characterizedaccordingly as described above.

Synthesis of NK1RL-SF5-Lys Peptide Linker-Rhodamine Conjugate

The purified NK1RL-SF5-Lys peptide linker (Compound 1 in FIG. 10A) wascoupled with NHS-rhodamine by stirring 1:1.2 ratios of the NK1RL-SF5-Lys peptide linker and NHS-rhodamine in dry DMSO, DIPEA underargon for 12 hours at room temperature. The resulting material waspurified by RP-HPLC (mobile phase A=20 raM ammonium acetate buffer, pH7, B=acetonitrile, gradient 10-100% B in 30 min, 13 mL/min, λ=280 nm).Pure fractions were combined, concentrated under vacuum, and lyophilizedto yield the product, NK1RL-SF5-Lys peptide linker-rhodamine conjugate(Compound 2 in FIG. 10A). The NK1RL-SF5-Lys peptide linker-rhodamineconjugate, a reddish solid, was analyzed using LC-MS and LR-ESIMS.

FIG. 10B shows the binding affinity of the NK1RL-SF5-Lys peptidelinker-rhodamine conjugate in cultured HEK 293-NK1R cells expressingNK-1 receptor.

Fluorescent Confocal Microscopy Imaging

HEK 293-NK1R cells (50,000 cells/well in 0.5 mL) were seeded intoconfocal microwell plate (Lab-Tek, Chambered #1.0 BorosilicateCoverglass) and allowed cells to form monolayers over 24 hours. Spentmedium was replaced with fresh medium containing NK1RL-SF5-Lys peptidelinker-rhodamine (25 nM) in the presence or absence of 100-fold excessfree ligand and cells were incubated for 1 hour at 37° C. After washingwith fresh medium (3×0.5 mL), confocal images were acquired using aconfocal microscopy (FV 1000, Olympus).

FIG. 10C shows resulting images from the binding studies of theNK1RL-SF5-Lys peptide linker-rhodamine conjugate to HEK 293-NK1R cells.Specifically, the confocal images provide (a) a magnification of cellsafter incubation for 1 hour at 37° C. in the absence of competing agent,(b) a magnified white light image of the cells in (a), (c) amagnification of the cells in the presence of a 100-fold excess ofcompeting agent (NK1RL alone) to the conjugate at 25 nM concentration,and (d) a magnified white light image of the cells in (c).

Example 7

Synthesis of NK1RL-SF5-Tyrosine peptide linker-S0456

A fluorescent imaging conjugate, NK1RL-SF5-tyrosine peptidelinker-S0456, was synthesized, as illustrated in FIG. 11, according tothe following steps.

Starting with a mixture of tert-butyl-L-tyrosine (1.1 equiv) andFmoc-PEG2-CO ₂H in CH2Cl2,1-[Bis(dimethylamino)methylene]-IH-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate (HATU) (1.2 equiv) was added, followed byDIPEA (5.0 equiv) under nitrogen. The reaction mixture was stirred for12 hours at room temperature. The reaction was then quenched with waterand 2% HCl solution, and extracted with EtOAc (3×5 mL). The combinedorganic layers were washed with brine, dried using sodium sulfate,filtered, and concentrated. The resulting residue was purified bysilica-gel column chromatography (hexane-EtOAc, 4:1) to afford a firstintermediary (Compound 1 in FIG. 11), which was confirmed by LC-MS.

Piperidine in THF (1:1) was added to the pure Compound I under nitrogenand stirred for 2 hours at room temperature. Completion of the reactionwas confirmed by LC-MS, and an excess of solvent and piperidine wereevaporated, resulting in a second intermediary (Compound 2 in FIG. 11).

Compound 2 (1.0 equiv) was mixed with NK1RL-SF5 (1.0 equiv) in CH₂Cl₂.HATU (1.2 equiv) was added to the mixture, followed by DIPEA (5.0 equiv)under nitrogen. The reaction mixture was stirred for 12 hours at roomtemperature. The reaction was quenched with water and 2% HCl solution,and extracted with EtOAc (3×5 mL). The combined organic layers werewashed with brine, dried with sodium sulfate, filtered, andconcentrated. The residue was purified by silica-gel columnchromatography (hexane:EtOAc, 4:1) to afford a third intermediary(Compound 3 in FIG. 11), which was confirmed by LC-MS.

S0456 dye (1.0 equiv) was added to the pure Compound 3 in DMF, followedby the addition of K2CO3 (5.0 equiv). The reaction mixture was stirredat 60° C. for 4 hours, and at room temperature for 12 hours undernitrogen. The progress of the reaction was monitored and confirmed byLC-MS. After completion of the reaction, crude mass was filtered toremove all solids, and filtrate was stirred with TFA for I hour at roomtemperature. The tert-butyl ester hydrolysis product was confirmed byLCMS and the crude mass was purified by RP-HPLC (mobile phase A=20 mMammonium acetate buffer, pH 7, B=acetonitrile, gradient 0-80% B in 30min, 13 mL/min, λ=280 nm). Pure fractions were combined, concentratedunder vacuum, and lyophilized to yield the product, NK1RL-SF5-tyrosinepeptide linker-S0456 (Compound 4 in FIG. 11). The product Compound 4 wasanalyzed by LC-MS and LR-ESIMS.

In Vitro Studies

HEK 293 NK1R cells (50,000 cells/well) were seeded into 24 well plates(BD Purecoat Amine, BD Biosciences) and allowed to grow to confluenceover 48-72 hours. Spent medium in each well was replaced with 0.5 mL offresh medium containing 0.5% bovine serum albumin and increasingconcentrations of NK1RL-SF5-Tyrosine peptide linker-S0456 in thepresence of 100-fold excess of competing ligand, i.e., L-733,060. Thebinding constant (Kd) was calculated by plotting fluorescence versus theconcentration of targeted radiotracer using GraphPad Prism 4 program,illustrated in FIG. 12.

Example 8

Synthesis of NK1RL-PEG2-DOTA Conjugate

DOTA-NHS ester (6.8 mg, 0.0135 mmol) was added to a purified NK1RL-PEG2linker (8 mg, 0.0135 mmol), followed by the addition of DIPEA in dryDMSO under argon. The reaction mixture was stirred for 12 hours at roomtemperature. The reaction progress was confirmed by LC-MS and purifiedby RP-HPLC (mobile phase A=20 mM ammonium acetate buffer, pH 7,B=acetonitrile, gradient 10-100% B in 30 min, 13 mL/min, λ=254 ran).Pure fractions were combined, concentrated under vacuum, and lyophilizedto yield the product, NK1 RL-PEG2-DOTA, was analyzed by LC-MS andLR-ESIMS.

In Vivo Studies

NK1R-transduced xenografts (HEK 293-NK1R) were prepared in groups, withthree mice per group. Doses of around 238 uCi of ligand per mouse wereintravenously administered, and the models were imaged at 4 hourspost-injection using PET, an example of which is shown in FIG. 13A. Theimages were analyzed in a region of interest (ROI) around the tumorxenograft activity, and percentage injected dose per mL (% ID/mL) valueswere calculated from the mean activity in the ROIs. FIG. 13B is a plotshowing this ROI activity for the NK1R-transduced xenografts. FIG. 13Cis a whole body mouse image on SPECT-CT for the NK1RL-PEG2-DOTA-1-11lnconjugate in a HEK 293-NK1R tumor xenograft model mouse.

The foregoing method descriptions and the process flow diagrams areprovided merely as illustrative examples and are not intended to requireor imply that the steps of the various embodiments must be performed inthe order presented. As will be appreciated by one of skill in the artthe order of steps in the foregoing embodiments may be performed in anyorder. Words such as “thereafter.” “then,” “next,” etc. are not intendedto limit the order of the steps; these words are simply used to guidethe reader through the description of the methods. Further, anyreference to claim elements in the singular, for example, using thearticles “a,” “an” or “the” is not to be construed as limiting theelement to the singular.

Skilled artisans may implement the above-described methods, processesand/or functionality in varying ways for each particular application,but such implementation decisions should not be interpreted as causing adeparture from the scope of the present invention.

The preceding description of the disclosed embodiments is provided toenable any person skilled in the art to make or use the presentinvention. Various modifications to these embodiments will be readilyapparent to those skilled in the art, and the generic principles definedherein may be applied to other embodiments without departing from thespirit or scope of the invention. Thus, the present invention is notintended to be limited to the embodiments shown herein but is to beaccorded the widest scope consistent with the following claims and theprinciples and novel features disclosed herein.

1-15. (canceled)
 16. A neurokinin-1 (NK-1) receptor-binding conjugatecomprising: an NK-1 receptor-binding moiety; an active agent comprisinga radionuclide; and a linker, which links the NK-1 receptor-bindingmoiety and the active agent, comprises at least one spacer linker, andoptionally further comprises a releasable linker.
 17. The NK-1receptor-binding conjugate of claim 16, wherein the active agent is aradio-imaging agent, an optical imaging agent, a position emissiontomography (PET)imaging agent, a magnetic resonance imaging (MRI)contrast agent, a computed tomography (CT) contrast agent, or afluorescence resonance energy transfer (FRET) imaging agent.
 18. TheNK-1 receptor-binding conjugate of claim 16, wherein the active agentcomprises a chelator in complex with a radionuclide selected from thegroup consisting of technetium-99m (^(99m)Tc), gallium-68 (⁶⁸Ga),indium-111 (¹¹¹In), yttrium-90 (⁹⁰Y), lutetium-177 (¹⁷⁷Lu), zirconium-89(⁸⁹Zr), actinium-225 (²²⁵Ac), cobalt-60 (⁶⁰Co), and copper-64 (⁶⁴Cu).19. The NK-1 receptor-binding conjugate of claim 16, wherein theradionuclide is ^(99m)Tc or ⁶⁴Cu.
 20. The NK-1 receptor-bindingconjugate of claim 18, wherein the chelator is selected from the groupconsisting of:

wherein each R is independently H, alkyl, heteroalkyl, cycloalkyl,heterocyclyl, alkenyl, alkynyl, aryl, heteroaryl, arylalkyl, orheteroarylalkyl, each of which is optionally substituted, and whereinone R comprises a heteroatom, which is attached to the linker; X isoxygen, nitrogen, or sulfur, and is attached to the linker; and n is aninteger from 1 to
 5. 21. The NK-1 receptor-binding conjugate of claim20, wherein the heteroatom in R is oxygen, nitrogen or sulfur.
 22. TheNK-1 receptor-binding conjugate of claim 19, wherein the chelator is atripeptide or tetrapeptide.
 23. The NK-1 receptor-binding conjugate ofclaim 22, wherein the tripeptide has the formula:

wherein each R is independently H, alkyl, heteroalkyl, cycloalkyl,heterocyclyl, alkenyl, alkynyl, aryl, heteroaryl, arylalkyl, orheteroarylalkyl, each of which is optionally substituted, and whereinone R comprises a heteroatom, which is attached to the linker.
 24. TheNK-1 receptor-binding conjugate of claim 23, wherein the heteroatom in Ris oxygen, nitrogen or sulfur.
 25. The NK-1 receptor-binding conjugateof claim 18, wherein the chelator is selected from the group consistingof:


26. The NK-1 receptor-binding conjugate of claim 16, wherein the NK-1receptor-binding moiety comprises a selective NK-1 receptor antagonistor a derivative thereof.
 27. The NK-1 receptor-binding conjugate ofclaim 16, wherein the NK-1 receptor-binding moiety is selected from thegroup consisting of:


28. The NK-1 receptor-binding conjugate of claim 16, wherein the spacerlinker comprises amino acids selected from the group consisting ofnaturally occurring amino acids and stereoisomers thereof.
 29. The NK-1receptor-binding conjugate of claim 16, wherein the linker comprisesdithioalkyloxycarbonyl.
 30. The NK-1 receptor-binding conjugate of claim16, wherein the linker comprises 3-thiosuccinimid-1-ylalkyloxy.
 31. TheNK-1 receptor-binding conjugate of claim 16, wherein the linkercomprises 3-cysteinylsuccinimid-1-ylalkyloxy, wherein the cysteinyl isoptically active or optically inactive.
 32. The NK-1 receptor-bindingconjugate of claim 16, wherein the conjugate is selected from:


33. A pharmaceutical composition comprising a NK-1 receptor-bindingconjugate of claim 16, and a pharmaceutically acceptable carrier,diluent, or excipient.
 34. A method of using a NK-1 receptor-bindingconjugate, which method comprises: (a) administering to a subject aneffective amount of the NK-1 receptor-binding conjugate of claim 16,optionally as a pharmaceutical composition comprising the NK-1receptor-binding conjugate and a pharmaceutically acceptable carrier,diluent, or excipient, and (b) detecting the radionuclide in thesubject.
 35. The method of claim 34, which further comprises diagnosinga disease, locating metastatic disease, detecting disease recurrence, ormonitoring response to therapy.
 36. The method of claim 35, whichfurther comprises selecting a patient for cholecystokinin 2 receptortargeted therapy.